An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4'-hydroxylated metabolites in breast cancer patients.

There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.

Determination of oseltamivir and oseltamivir carboxylate in human plasma by liquid chromatography-tandem mass spectrometry

Introduction: Oseltamivir phosphate (OP), the prodrug of oseltamivir carboxylate (OC; active metabolite), is marketed since 10 years for the treatment of seasonal influenza flu. It has recently received renewed attention because of the threat of avian flu H5N1 in 2006-7 and the 2009-10 A/H1N1 pandemic. However, relatively few studies have been published on OP and OC clinical pharmacokinetics. The disposition of OC and the dosage adaptation of OP in specific populations, such as young children or patients undergoing extrarenal epuration, have also received poor attention. An analytical method was thus developed to assess OP and OC plasma concentrations in patients receiving OP and presenting with comorbidities or requiring intensive care. Methods: A high performance liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) requiring 100-µL aliquot of plasma for quantification within 6 min of OP and OC was developed. A combination of protein precipitation with acetonitrile, followed by dilution of supernant in suitable buffered solvent was used as an extraction procedure. After reverse phase chromatographic separation, quantification was performed by electro-spray ionization-triple quadrupole mass spectrometry. Deuterated isotopic compounds of OP and OC were used as internal standards. Results: The method is sensitive (lower limit of quantification: 5 ng/mL for OP and OC), accurate (intra-/inter-assay bias for OP and OC: 8.5%/5.5% and 3.7/0.7%, respectively) and precise (intra-/inter-assay CV%: 5.2%/6.5% and 6.3%/9.2%, respectively) over the clinically relevant concentration range (upper limits of quantification 5000 ng/mL). Of importance, OP, as in other previous reports, was found not to be stable ex vivo in plasma on standard anticoagulants (i.e. EDTA, heparin or citrate). This poor stability of OP has been prevented by collecting blood samples on commercial fluoride/oxalate tubes. Conclusions: This new simple, rapid and robust HPLC-MS/MS assay for quantification of OP and OC plasma concentrations offers an efficient tool for concentration monitoring of OC. Its exposure can probably be controlled with sufficient accuracy by thorough dosage adjustment according to patient characteristics (e.g. renal clearance). The usefulness of systematic therapeutic drug monitoring in patients appears therefore questionable. However, pharmacokinetic studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

Approximate knowledge of rationality and correlated equilibria

We extend Aumann's theorem [Aumann 1987], deriving correlated equilibria as a consequence of common priors and common knowledge of rationality, by explicitly allowing for non-rational behavior. Wereplace the assumption of common knowledge of rationality with a substantially weaker one, joint p-belief of rationality, where agents believe the other agents are rational with probability p or more. We show that behavior in this case constitutes a kind of correlated equilibrium satisfying certain p-belief constraints, and that it varies continuously in the parameters p and, for p sufficiently close to one,with high probability is supported on strategies that survive the iterated elimination of strictly dominated strategies. Finally, we extend the analysis to characterizing rational expectations of interimtypes, to games of incomplete information, as well as to the case of non-common priors.

Discrete devaluations and multiple equilibria in a first generation model of currency crises

The first generation models of currency crises have often been criticized because they predict that, in the absence of very large triggering shocks, currency attacks should be predictable and lead to small devaluations. This paper shows that these features of first generation models are not robust to the inclusion of private information. In particular, this paper analyzes a generalization of the Krugman-Flood-Garber (KFG) model, which relaxes the assumption that all consumers are perfectly informed about the level of fundamentals. In this environment, the KFG equilibrium of zero devaluation is only one of many possible equilibria. In all the other equilibria, the lack of perfect information delays the attack on the currency past the point at which the shadow exchange rate equals the peg, giving rise to unpredictable and discrete devaluations.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

On equilibria in duopolies with finite strategy spaces

We will call a game a reachable (pure strategy) equilibria game if startingfrom any strategy by any player, by a sequence of best-response moves weare able to reach a (pure strategy) equilibrium. We give a characterizationof all finite strategy space duopolies with reachable equilibria. Wedescribe some applications of the sufficient conditions of the characterization.

Existence of sparsely supported correlated equilibria

We show that every finite N-player normal form game possesses a correlated equilibrium with a precise lower bound on the number of outcomes to which it assigns zero probability. In particular, the largest games with a unique fully supported correlated equilibrium are two-player games; moreover, the lower bound grows exponentially in the number of players N.

Contracting externalities and multiple equilibria in sectors: Theory and evidence

We consider an economy where the production technology has constantreturns to scale but where in the descentralized equilibrium thereare aggregate increasing returns to scale. The result follows froma positive contracting externality among firms. If a firms issurrounded by more firms, employees have more opportunitiesoutside their own firm. This improves employees' incentives toinvest in the presence of ex post renegotiation at the firm level,at not cost. Our leading result is that if a region is sparselypopulated or if the degree of development in the region is lowenough, there are multiple equilibria in the level of sectorialemployment. From the theoretical model we derive a non-linearfirst-order censored difference equation for sectoral employment.Our results are strongly consistent with the multiple equilibriahypothesis and the existence of a sectoral critical scale (belowwich the sector follows a delocation process). The scale of theregions' population and the degree of development reduce thecritical scale of the sector.

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography.

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid-liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R- didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated 3-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.

A note on the convergence to competitive equilibria in economies with moral hazard

We examine the conditions under which competitive equilibria can beobtained as the limit, when the number of strategic traders getslarge, of Nash equilibria in economies with asymmetric informationon agents' effort and possibly imperfect observability of agents'trades. Convergence always occur when either effort is publiclyobserved (no matter what is the information available tointermediaries on agents' trades); or effort is private informationbut agents' trades are perfectly observed; or no information at allis available on agents' trades. On the other hand, when eachintermediary can observe its trades with an agent, but not theagent's trades with other intermediaries, the (Nash) equilibriawith strategic intermediaries do not converge to any of thecompetitive equilibria, for an open set of economies. The source ofthe difficulties for convergence is the combination of asymmetricinformation and the restrictions on the observability of tradeswhich prevent the formation of exclusive contractual relationshipsand generate barriers to entry in the markets for contracts.

A simple model of multiple equilibria based on risk

This paper shows how risk may aggravate fluctuations in economies with imperfect insurance and multiple assets. A two period job matching model is studied, in which risk averse agents act both as workers and as entrepreneurs. They choose between two types of investment: one type is riskless, while the other is a risky activity that creates jobs.Equilibrium is unique under full insurance. If investment is fully insured but unemployment risk is uninsured, then precautionary saving behavior dampens output fluctuations. However, if both investment and employment are uninsured, then an increase in unemployment gives agents an incentive to shift investment away from the risky asset, further increasing unemployment. This positive feedback may lead to multiple Pareto ranked equilibria. An overlapping generations version of the model may exhibit poverty traps or persistent multiplicity. Greater insurance is doubly beneficial in this context since it can both prevent multiplicity and promote risky investment.

Stable sunspot equilibria in a cash-in-advance economy

We analyze a monetary model with flexible labor supply, cash-inadvance constraints and seigniorage-financed government deficits. If the intertemporal elasticity of substitution of labor is greater than one, there are two steady states, one determinate and the other indeterminate. If the elasticity is less than one, there is a unique steady state, which can be indeterminate. Only in the latter case do there exist sunspot equilibria that are stable under adaptive learning. A sufficient reduction in government purchases can in many cases eliminate the sunspot equilibria while raising consumption/labor taxes even enough to balance the budget may fail to achieve determinacy.